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Journal of Neuroinflammation
Open Access
Review
The microglial NADPH oxidase complex as a source of oxidative
stress in Alzheimer's disease
Brandy L Wilkinson* and Gary E Landreth
Address: Alzheimer Laboratory, Department of Neuroscience, Case Western Reserve University, Cleveland, OH 44106, USA
Email: Brandy L Wilkinson* - ; Gary E Landreth -
* Corresponding author
Abstract
Alzheimer's disease is the most common cause of dementia in the elderly, and manifests as
progressive cognitive decline and profound neuronal loss. The principal neuropathological
hallmarks of Alzheimer's disease are the senile plaques and the neurofibrillary tangles. The senile
plaques are surrounded by activated microglia, which are largely responsible for the
proinflammatory environment within the diseased brain. Microglia are the resident innate immune
cells in the brain. In response to contact with fibrillar beta-amyloid, microglia secrete a diverse array
of proinflammatory molecules. Evidence suggests that oxidative stress emanating from activated
microglia contribute to the neuronal loss characteristic of this disease. The source of fibrillar beta-
amyloid induced reactive oxygen species is primarily the microglial nicotinamide adenine
dinucleotide phosphate (NADPH) oxidase. The NADPH oxidase is a multicomponent enzyme
complex that, upon activation, produces the highly reactive free radical superoxide. The cascade of
intracellular signaling events leading to NADPH oxidase assembly and the subsequent release of
superoxide in fibrillar beta-amyloid stimulated microglia has recently been elucidated. The
induction of reactive oxygen species, as well as nitric oxide, from activated microglia can enhance
the production of more potent free radicals such as peroxynitrite. The formation of peroxynitrite
causes protein oxidation, lipid peroxidation and DNA damage, which ultimately lead to neuronal
cell death. The elimination of beta-amyloid-induced oxidative damage through the inhibition of the
NADPH oxidase represents an attractive therapeutic target for the treatment of Alzheimer's

disease.
Background
Alzheimer's disease (AD) is the most common form of
senile dementia and is characterized by progressive cogni-
tive impairment and profound neuronal loss. The neu-
ropathological hallmarks of AD are the senile plaques
consisting of extracellular deposits of fibrillar β-amyloid
(fAβ) and the intracellular neurofibrillary tangles com-
posed of hyperphosphorylated tau. While the events lead-
ing to AD onset remain elusive, the progression of disease
pathology has been thoroughly examined and several
mechanisms of neuronal damage have been identified.
Considerable attention has been focused on the role of
inflammatory mechanisms in the etiology of AD, and
senile plaques are the site of local inflammatory response
[1-3]. Epidemiological studies also provide persuasive evi-
dence for the involvement of inflammatory mechanisms
in AD. Patients using nonsteroidal anti-inflammatory
drugs (NSAIDs) were found to have a dramatically
Published: 09 November 2006
Journal of Neuroinflammation 2006, 3:30 doi:10.1186/1742-2094-3-30
Received: 25 September 2006
Accepted: 09 November 2006
This article is available from: />© 2006 Wilkinson and Landreth; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( />),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Journal of Neuroinflammation 2006, 3:30 />Page 2 of 12
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reduced incidence of disease. These studies reported that
patients treated with NSAIDs for a 2-year period or more

had as much as 60–80% reduction in the risk for AD [4-
6]. Long-term NSAID treatment attenuated disease onset,
slowed the rate of cognitive impairment and reduced the
level of overall symptomatic severity [6,7]. In both
humans and mice, NSAID treatment is associated with
reduced Aβ plaque burden and a reduction in plaque-
associated microglia [8-11]. NSAIDs have also been
shown to exhibit pleiotropic effects on the processing of
the amyloid precursor protein (APP) [12-14]. These data
support the potential utility of anti-inflammatory drug
therapies in the treatment of AD.
Microglia are the principle immune effector cells in the
brain and represent approximately 5–10% of all glia
found in the brain. The density of microglia in the brain is
approximately 6 × 10
3
cells/mm
3
[15]. Microglia are
derived from a myeloid lineage, and originate from bone
marrow-derived progenitor cells that are trafficked from
the periphery into the brain parenchyma where they dif-
ferentiate [16-19]. It has recently been appreciated that
"resting" or "quiescent" microglia are highly dynamic and
constantly extend their processes to survey their microen-
vironment. This surveillance permits quick reaction to
either local injury or invading pathogens [15,20].
In the AD brain, microglia are characterized by a reactive
phenotype and are found surrounding senile plaques
[21]. These cells mount a local inflammatory response

and express cell surface receptors reflective of the pheno-
typic activation of this cell type [22]. Specifically, activated
microglia secrete inflammatory cytokines such as tumor
necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β),
IL-6, and chemokines, all of which are found at elevated
levels in the AD brain [3]. Fibrillar Aβ-stimulated micro-
glia also release reactive oxygen species (ROS) and reac-
tive nitrogen intermediates (RNI) [3]. A chronic
microglial inflammatory response leads to the continued
release of inflammatory mediators that are associated
with neurotoxic injury to surrounding neurons (Goerdt
and Orfanos 1999; Wyss-Coray and Mucke 2002).
In the CNS, oxidative damage generally manifests as lipid
peroxidation and the formation of protein oxidation
products that are toxic to neurons. Neurons are inherently
susceptible to oxidative damage because of high respira-
tory turnover, dependency on oxidative phosphorylation
reactions, high concentrations of catalytic iron and low
levels of antioxidant defense enzymes [23]. Oxidative
damage is observed early in the progression of AD
[24,25], and can be detected prior to fAβ deposition both
in the human brain [26] and animal models of the disease
[24]. These findings suggest that oxidative damage ema-
nating from the reactive microglia and astrocytes adjacent
to senile plaques may play an early role in the pathogene-
sis of AD.
Several potential sources of ROS exist within microglia
and astrocytes including the nicotinamide adenine dinu-
cleotide phosphate (NADPH) oxidase, mitochondria res-
piratory chain, xanthine oxidase, microsomal enzymes,

cycloxygenase and lipoxygenase. In response to fAβ; how-
ever, it is believed that the primary source of ROS and the
source of widespread oxidative damage found in both AD
brains and mouse models of AD is the microglial NADPH
oxidase [26-30].
Despite ample evidence supporting microglial NADPH
oxidase participation in fAβ-stimulated ROS production
and oxidative damage, until recently little was known
about the signaling pathway(s) subserving NADPH oxi-
dase assembly. A mechanistic linkage between Aβ fibril
engagement of the cell surface receptor complex and the
initiation of intracellular signaling events regulating oxi-
dase assembly and activation has been described [31,32].
The NADPH oxidase
The phagocytic NADPH oxidase plays an essential role in
innate immunity by catalyzing the formation of superox-
ide (O
2
-
), which facilitates the destruction of invading
microorganisms during phagocytosis. Upon oxidase acti-
vation, O
2
-
is produced and participates in microbial kill-
ing. However, other more potent ROS are rapidly formed
including hydrogen peroxide, hydroxyl radical, peroxyni-
trite and other oxidants [33]. The excessive production of
these free radicals can damage tissue adjacent to the sites
of inflammatory action; therefore, the activation of the

NADPH oxidase is tightly controlled though regulated
assembly of the individual oxidase subunits into a func-
tionally active complex [34].
The NADPH oxidase consists of two integral membrane
proteins, p22
phox
and gp91
phox
, which together form a het-
erodimeric flavoprotein known as cytochrome b
558
(Fig-
ure 1). In addition, there are four cytosolic components
p47
phox
, p67
phox
, p40
phox
, and the small G-protein Rac
(Figure 1). Activation of the NADPH oxidase occurs when
the macrophage/microglial cell is exposed to inflamma-
tory stimuli initiating the activation of multiple parallel
intracellular signaling cascades. These cytoplasmic signal-
ing events stimulate the phosphorylation of p47
phox
and
p67
phox
and the GDP/GTP exchange on Rac. The cytosolic

components then translocate to the membrane where
they form a complex with cytochrome b
558
. The oxidase
complex then initiates electron flow and generation of O
2
-
through the NADPH-derived electron reduction by the
flavocytochrome (Figure 1).
Journal of Neuroinflammation 2006, 3:30 />Page 3 of 12
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In recent years, it has become evident that several different
forms of the NADPH oxidase exist and comprise a family
of related enzymes, which are differentially expressed in a
variety of tissue types. While the presence of these various
subunits in different tissue types remains under debate, it
is clear that ROS function more broadly than as simply
one consequence of the immunological response. The
defining element of these enzyme complexes is the cata-
lytic gp91 subunit. A new nomenclature has now been
applied to this family of enzymes with classification based
on the structure of the gp91 subunit. The classical phago-
cytic NADPH oxidase incorporates the gp91 subunit and
is termed NOX2 [35].
Cytosolic p47
phox
and p67
phox
are often found as a com-
plex and both require phosphorylation prior to their

translocation to the membrane [34,36]. Analysis of
p47
phox
and p67
phox
phosphorylation has been investi-
gated with the use of specific protein kinase inhibitors
revealing that protein kinase C (PKC) [37-39], p21-acti-
vated kinase-1 (PAK1) [40], mitogen-activated protein
kinase (MAPK) [41], Akt [42], and phosphatidylinositol-
3 kinase (PI3K) [43] phophorylate p47
phox
or p67
phox
. The
diversity of kinases implicated in the phosphorylation of
p47
phox
and p67
phox
suggests that the intracellular signal-
ing pathways responsible for this phosphorylation event
are complex and may be cell type and stimulus specific.
There are several isoforms of the Rac GTPase, with Rac1
being the predominant isoform in macrophages and
monocytic cells while Rac2 is predominantly expressed in
neutrophils [44]. Rac is a member of the Rho-family of
small monomeric GTPases. Like all Ras-superfamily mem-
bers, Rac1 acts as a molecular switch cycling between an
inactive guanosine diphosphate (GDP)-bound state and

an active guanosine triphosphate (GTP)-bound state that
can bind target proteins (Figure 2). This process is facili-
tated by a group of molecules known as guanine-nucle-
otide exchange factors (GEFs). In resting cells, Rac is
anchored in the cytosol through an interaction between
its C-terminal prenyl moiety and the GDP dissociation
inhibitor, RhoGDI. During activation by an inflammatory
Activation of the phagocytic NADPH oxidase complexFigure 1
Activation of the phagocytic NADPH oxidase complex. Stimulation of the phagocyte induces the parallel activation of oxidase
components within the cytoplasm. This activation causes the conversion of Rac into an active GTP-bound form and the phos-
phorylation of p47
phox
and p67
phox
. These subunits then translocate to the membrane where they interact with p22
phox
and
gp91
phox
(NOX2) to initiate reactive oxygen production.
Journal of Neuroinflammation 2006, 3:30 />Page 4 of 12
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stimulus, Rac binds GTP and dissociates from RhoGDI.
Rac is then able to translocate to the plasma membrane,
where its prenyl group inserts into the membrane, tether-
ing Rac to its inner face and facilitating Rac's interaction
with p67
phox
[45]. While the precise role of Rac in NADPH
oxidase activation is not fully understood, several lines of

evidence suggest Rac acts as an adaptor molecule for
p67
phox
[46,47], and may be actively involved in the elec-
tron transfer process itself [48,49]. The requirement for
Rac-GTP has been further established through in vitro
studies demonstrating that the addition of dominant-neg-
ative Rac or overexpression of Rho-GDI severely dimin-
ishes superoxide production [50,51].
Microglia and the NADPH oxidase
We have recently described a multireceptor cell surface
complex for fAβ on microglia [32]. Microglial contact
with fAβ catalyzes the assembly of an ensemble of cell sur-
face receptors that includes CD36, α
6
β
1
integrin, CD47,
and the class A scavenger receptor (SRA) (Figure 3). Fibril-
lar Aβ engagement of this receptor complex leads to the
initiation of complex signaling events leading to protein
tyrosine phosphorylation and activation of the src-family
kinases Lyn and Fyn as well as the tyrosine kinase Syk
[30,32,52]. Activation of these signaling cascades are
linked to the synthesis and secretion of proinflammatory
molecules and cytokines [28,30,52-58].
Microglia exposed to fAβ exhibit a respiratory burst lead-
ing to the release of superoxide anion [28,30,32,52,59].
Release of ROS is mediated through the fAβ cell surface
receptor complex [31,32]. Furthermore, global inhibition

of src-family tyrosine kinases or inhibition of phosphati-
dylinositol-3 kinase (PI3K) attenuates ROS production.
These findings suggest that these kinases are involved in
upstream signaling cascades responsible for activating
NADPH oxidase assembly in response to fAβ peptides
[28,30].
In both human AD brain tissue [27] and fAβ-treated cul-
tured monocytes and primary microglia [28], there is a
translocation of both the p47
phox
and p67
phox
subunits
from the cytosol to the membrane. Fibrillar Aβ-stimula-
tion also results in a relative increase in mRNA transcripts
for both p47
phox
and gp91
phox
and an increase in p47
phox
protein expression in monocytes primed with INFγ [59].
Activation of the Rac GTPaseFigure 2
Activation of the Rac GTPase. GTP-binding of the Rac protein confers an "active" confirmation allowing Rac to modulate
downstream effectors. Rac is regulated by upstream effectors know as GEFs which facilitate the exchange of GDP for GTP and
increase the amount of active Rac.
Journal of Neuroinflammation 2006, 3:30 />Page 5 of 12
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Recently, we have demonstrated that the Rac GTPase is
also activated and subsequently translocated from the

cytosol to the membrane of THP-1 monocytes in a fAβ-
dependent manner [31]. A detailed examination of the
mechanisms subserving oxidase activation has revealed
that upon fAβ stimulation, the Vav guanine nucleotide
exchange factor (GEF) is a key modulator of NADPH oxi-
dase assembly in monocytes and primary microglia (Fig-
ure 3). Vav is responsible for the exchange of GDP for GTP
on the Rac GTPase. In order for Vav to exert its GEF activ-
ity, it must be tyrosine phosphorylated [60]. The tyrosine
phosphorylation of Vav requires fibril engagement of the
Aβ cell surface receptor complex as well as activation of
tyrosine kinase cascades involving Lyn or Syk [31]. Impor-
tantly, genetic deletion of Vav from primary microglia
resulted in severe attenuation of ROS production follow-
ing fAβ treatment [31].
Confirmation of NADPH oxidase participation in Aβ-
induced ROS production has been obtained utilizing cells
obtained from patients with the rare recessive genetic dis-
order, chronic granulomatous disease (CGD). This dis-
ease is characterized by a mutation in the genes that
encode one of the essential subunits of the NADPH oxi-
dase: p22
phox
, p47
phox
, p67
phox
or gp91
phox
. These defects

render the cells unable to generate H
2
O
2
in response to
any agonist of the oxidase. Bianca and colleagues showed
that monocytes and neutrophils from CGD patients fail to
Model of intracellular signaling following Aβ fibril interaction with the microglial cell surface receptor complexFigure 3
Model of intracellular signaling following Aβ fibril interaction with the microglial cell surface receptor complex. Fibrillar engage-
ment of an ensemble of cell surface receptors initiates a tyrosine kinase-based signaling cascade. Tyrosine phosphorylation of
the Vav-GEF results in the activation of downstream Rac1 GTPase.
Journal of Neuroinflammation 2006, 3:30 />Page 6 of 12
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produce ROS in response to fAβ peptides or to phorbol
12-myristate 13-acetate (PMA) [28]. Subsequent studies
have substantiated that the NADPH oxidase is essential
for Aβ-induced ROS production. Elegant in vivo data from
Park and colleagues assessed ROS production in the neo-
cortex using hydroethidine fluoromicrography [61].
Fibrillar Aβ superfused through a cranial window
increased ROS production in the neocortex. This effect
could be abolished with the addition of a peptide inhibi-
tor to the gp91
phox
subunit. These authors further demon-
strated that ROS levels were increased in the Tg2576
mouse model of AD; however, no signs of ROS produc-
tion were evident in a mouse model where the Tg2576
mouse lacked the gp91
phox

gene.
Surprisingly, the contribution of ROS to fAβ-stimulated
neurotoxicity has not been extensively examined. Previ-
ous studies have reported that proinflammatory mole-
cules secreted from fAβ-stimulated microglia lead to
neuronal apoptosis [52,62]. However, it has only recently
been appreciated that in a microglia/APP-expressing neu-
roblastoma cell co-culture model inhibition of ROS activ-
ity with superoxide dismutase or catalase (ROS
scavengers) resulted in decreased neuronal cell death [63].
These authors validated their findings using a NADPH
oxidase-deficient (gp91
phox
null macrophage cell line in a
similar co-culture model. The oxidase-deficient cells were
unable to kill the APP-expressing neuroblastoma cells.
Taken together, these findings argue that the interaction of
Aβ with microglia and the assembly of the active micro-
glial NADPH oxidase maybe largely responsible for the
oxidative damage observed in the AD brain.
Astrocytes and the NADPH oxidase
Astrocytes are the most abundant glial cell type in the
brain and greatly outnumber microglia [64]. Historically,
astrocytes were believed to function solely as supporting
cells; however, this dogma has recently undergone sub-
stantial re-evaluation. Astrocytes are also involved with
synaptic efficacy, neurogenesis, gliogenesis and even
inflammatory processes [65,66]. In the AD brain, it has
been postulated that the abundance of astrocytes results
in their extensive exposure to Aβ from the earliest stages of

the disease. Indeed, reactive astrocytes are found adjacent
to senile plaques. Plaque-associated astrocytes upregulate
the expression of the chemokines MCP-1 and RANTES,
which act as chemoattractants for microglia, These cells
also release the proinflammatory cytokines TNFα, IL1β, as
well as nitric oxide (NO) [67].
The existence of an astrocytic NADPH oxidase is contro-
versial. Shimohama and colleagues examined microglia,
neurons and astrocytes in culture for the presence of either
p67
phox
or p47
phox
, and these oxidase subunits were not
detected in astrocytes [27]. Additionally, in a co-culture
model including neuron-enriched, microglia-enriched,
and astrocyte-enriched cultures, the addition of fAβ failed
to stimulate the generation of ROS in all cultures except
the microglia [68]. These data support the prevailing view
that astrocytes are not a significant source of ROS. How-
ever, recent findings have challenged this hypothesis.
Abramov et al. have reported that astrocytes express the
full complement of NADPH oxidase subunits [69]. In pri-
mary astrocytes cultures, NADPH oxidase activation was
induced by exposure to fAβ peptides and this response
was abolished by the addition of the NADPH oxidase
inhibitors diphenylene iodonium (DPI), apocynin, or 4-
(2-aminoethyl)-benzene-sulphonylfluoride [29,70]. Cur-
rently, the receptors and signaling pathways responsible
for fAβ-dependent NADPH oxidase complex formation in

astrocytes remain unknown. Unlike the fAβ multireceptor
cell surface complex described in microglia, astrocytes
must initiate complex formation differently in response to
fAβ as preincubation of cells with a CD36 blocking anti-
body had no effect on fAβ-induced ROS generation [70].
The fAβ induced ROS production in astrocytes is postu-
lated to be a consequence of the loss of mitochondrial
membrane potential and glutathione depletion; however,
exactly how fAβ activates the astrocytic NADPH oxidase
remains unclear [29,70].
Abramov and colleagues have examined a potential role
for astrocytic NADPH oxidase-derived ROS in fAβ-
dependent neuronal death. In an astrocyte/neuron co-cul-
ture model, fAβ treatment caused cell death in both cell
types but to a much larger extent in neurons. This
response was almost completely reversed by treatment
with the NADPH oxidase inhibitor DPI [29,70]. Moreo-
ver, treatment with antioxidants and glutathione precur-
sors also decreased the neurotoxicity [29,70]. However, it
remains uncertain how the astrocytes promote neuronal
cell death.
Neurons and the NADPH oxidase
Neuronal NADPH oxidase activity has not been widely
examined. The presence of phagocytic NADPH oxidase
(NOX2) subunits within cultured cortical and sympa-
thetic neurons has been reported [71,72]. It has also
recently been appreciated that the NOX4 subunit is
present in mouse brain in cortex, in hippocampal neu-
rons, and in Purkinje cells [73]. Neuronal cells respond to
toxic insults including ischemia [73], zinc overload [72],

and nerve growth factor deprivation [71] by inducing the
expression and translocation of NADPH oxidase subunits.
In the central nervous system, Noh and Koh (2000) were
able to demonstrate increased NADPH oxidase-derived
(NOX2) ROS production in cortical cultures in response
to zinc exposure [72]. In response to fAβ peptides, how-
ever, neurons fail to generate an NADPH oxidase derived
respiratory burst [68]. These findings suggest that the pres-
Journal of Neuroinflammation 2006, 3:30 />Page 7 of 12
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ence of NADPH oxidase complex subunits within neu-
rons may mediate signaling pathways that regulate some
other aspect of cellular response [74].
Role of the NADPH oxidase in peroxynitrite
formation and oxidative damage
There is substantial evidence supporting the fAβ-induced
production of superoxide from the NADPH oxidase in
microglia. In addition, neurons, microglia and astrocytes
are capable of generating substantial amounts of nitric
oxide (NO) through the inducible nitric oxide synthase
(iNOS) [75-77]. Fibrillar Aβ peptides stimulate the induc-
tion of iNOS and NO production through an NADPH-
dependent oxidative deamination of L-arginine
[56,75,78,79]. In vitro studies have demonstrated that
monocytes, microglia, and astrocytes release NO in
response to fAβ and this response is enhanced by the
proinflammatory cytokines [56,75,80]. Microglia/neuron
co-culture studies reveal that the NO released from Aβ-
stimulated microglia is toxic to neurons, and this effect is
exacerbated by the addition of INFγ [81]. NO production

is necessary for neuronal cell death as inhibition of nitrite
production with a NO synthase inhibitor attenuates NO-
induced neurotoxicity [81,82].
Inflammation-induced activation of microglial NADPH
oxidase and iNOS has been reported to act synergistically
to kill neurons through the formation of peroxynitrite
[82]. Peroxynitrite is a potent oxidant with biological reac-
tivity similar to that of the hydroxyl radical [83]. NO and
superoxide form peroxynitrite at nearly the diffusion con-
trolled rate [k = 6.7 × 10
9
M
-1
s
-1
] [84]. Peroxynitrite pro-
motes the tyrosine nitration and nitrosylation of cysteines
within cellular proteins. The addition of nitrite to tyrosine
residues is extremely detrimental as it leads to both pro-
tein and enzyme dysfunction and eventual death of cul-
tured neurons [82,85-87]. Peroxynitrite can directly
oxidize proteins [88], lipids [89] and DNA [90].
Peroxynitrite is postulated to be associated with AD
pathogenesis [86,91-94]. Levels of both dityrosine and
nitrotyrosine are elevated in brain regions specifically
affected in AD [91]. Protein nitration is also increased in
the hippocampus of AD patients compared to age-
matched controls, and nitrotyrosine is evident in, but not
restricted to, neurons containing neurofibrillary tangles
[92]. Recent advances in proteomics have also identified

specific proteins which are nitrated in the AD brain
including β-actin, enolase, and L-lactate dehydrogenase
[94].
The role microglia play in peroxynitrite-mediated neuro-
toxicity has only recently been described. Aβ-mediated
neuronal apoptosis in vitro is likely a consequence of the
microglial secretion of proinflammatory cytokines includ-
ing TNFα [3,95]. Importantly, TNFα stimulates increased
iNOS in neuronal cells [62,96,97]. Combs et al. (2001)
demonstrated that conditioned media from Aβ-stimu-
lated primary microglia produce a TNFα/iNOS-depend-
ent neuronal apoptosis, which could be rescued with the
addition of either a TNFα antibody or a iNOS-selective
inhibitor [62]. Levels of both intracellular neuronal NO
and nitrotyrosine, a marker or peroxynitrite activity, were
elevated by the addition of Aβ-conditioned media. An
iNOS selective inhibitor eliminated the levels of both
molecules [62]. Similarly, the addition of a peroxynitrite
decomposition catalyst (FeTM PyP) blocked the neurotox-
icity of Aβ-stimulated microglia, and a superoxide dis-
mutase (SOD) mimetic (MnTM PYP), which blocks
peroxynitrite formation by scavenging superoxide radi-
cals, also ameliorates neuronal cell death [98]. Taken
together, these data suggest that Aβ-stimulated produc-
tion of peroxynitrite plays an important role in the patho-
genesis of oxidative damage in the AD brain.
Oxidative damage directly stemming from peroxynitrite
formation has been examined in the AD brain. Immuno-
cytochemical 4-hydroxynonenal (HNE) analysis demon-
strated increased lipid peroxidation in AD brain tissue

[99,100]. Using 8-hydroxyguanine (8-OHdG), a widely
studied biomarker for DNA oxidative damage, several
groups have also demonstrated increased nucleic acid
damage in the AD brain [101,102]. However, a direct link-
age between these oxidative events and the microglial
NADPH oxidase has not been established.
Antioxidant therapy and the NADPH oxidase
Considerable attention has been devoted to antioxidants
as a potential therapeutic intervention in AD. These com-
pounds reduce oxidative stress by scavenging free radicals.
A large number of candidate antioxidants exist including
the monophenolic compounds: tocopherol, 17β-estra-
diol, and 5-hydroxytryptamine (serotonin) and the
polyphenolic compounds: flavonoids, stilbenes, and hyd-
roquinones [103]. Among these potential antioxidants, α-
tocopherol (vitamin E) has been the most widely studied
for the treatment of AD.
While previous studies have not directly analyzed the rela-
tionship between vitamin E supplementation and fAβ-
dependent activation of the NADPH oxidase, there is evi-
dence suggesting vitamin E could potentially inhibit an
Aβ-induced respiratory burst. First, microglial cells treated
with vitamin E transition into a "resting" morphological
phenotype and downregulation the expression of adhe-
sion molecules [104]. Second, in vitro studies demonstrate
that BV-2 murine microglia or primary microglial pre-
incubated with vitamin E have an attenuated respiratory
burst in response to PMA. These cells also exhibit defects
in the phosphorylation and translocation of p47
phox

[105]
Journal of Neuroinflammation 2006, 3:30 />Page 8 of 12
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and p67
phox
[106] to the membrane. Third, vitamin E
treatment decreases NO production and induction of
TNFα and IL-1α in lipopolysaccaride (LPS)-stimulated
microglial cells [107]. Early vitamin E supplementation
also decreases lipid peroxidation [108]. Finally, vitamin E
imparts a neuroprotective effect on neuronal cells in
microglia-neuron cocultures stimulated with LPS [107].
Taken together, these findings provide compelling in vitro
evidence that vitamin E could act to inhibit the Aβ-stimu-
lated microglia inflammatory response. Specifically, vita-
min E could inhibit ROS and RNI production.
In a transgenic mouse model of AD, dietary vitamin E
reduced the formation of Aβ
1–40
and Aβ
1–42
and also
reduced Aβ plaque burden in the brains of young but not
aged Tg2576 mice [108]. The efficacy of dietary vitamin E
has also been studied in clinical trials with patients with
moderately severe AD. Daily treatment with vitamin E
slowed the progression of the disease, but did not
improve cognitive scores over a two-year period [109].
Zandi and colleagues more recently analyzed the relation-
ship between antioxidant supplements and AD risk, and

concluded that use of vitamin E and vitamin C in combi-
nation reduced the prevalence and incidence of AD in an
elderly population; however, the use of either of these
vitamins alone had no effect [110]. Finally, in patients
diagnosed with mild cognitive impairment (MCI), a tran-
sitional state between normal brain aging and AD, daily
vitamin E supplementation failed to prolong the rate of
progression to AD [111]. These studies suggest that early
dietary supplementation with vitamin E in combination
with additional antioxidants may impart a protective
effect against AD onset; however, it remains unclear how
the effects of antioxidant treatment are mediated. Little
attention has been focused on how antioxidant therapy
might affect the proinflammatory responses of microglia
or astrocytes to Aβ peptides.
Statin therapy and the NADPH oxidase
The rationale for statin therapy as a potential treatment
for AD arose from several epidemiological studies, which
reported that hypercholesterolemia in midlife could pre-
dict the later occurrence of AD [112,113]. As a therapeutic
agent, the primary action of statins is to competitively
inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-
CoA) reductase the rate limiting enzyme in the cholesterol
biosynthetic pathway. Statins, therefore, block the de novo
synthesis of cholesterol and ultimately lead to reduced
plasma cholesterol levels. In addition to their ability to
lower low-density lipoproteins, statins exhibit additional
pleiotropic effects including modulation of inflammatory
processes. The anti-inflammatory actions of statins are
believed to arise from their ability to prevent the synthesis

of isoprenoid intermediates that are responsible for the
posttranslational addition of lipid attachments on the c-
terminus of a variety of proteins including small GTPases
such as Rac. The protein isoprenylation of Rac is critical
for Rac's subcellular localization, interactions with
RhoGDI, anchoring to the plasma membrane and ulti-
mately to the activation of inflammatory signaling path-
ways [114].
In vitro evidence suggests the pleiotropic effects of statin
treatment suppresses the microglia proinflammatory
response to Aβ fibrils. Statin pretreatment abolished the
fAβ-stimulated production of ROS in monocytes [115]. In
addition, treatment of monocytes and microglia with sim-
vastatin has been shown to uncouple the interaction
between Rac and its negative regulator Rho-GDI and also
disrupts Rac's ability to interact with the plasma mem-
brane [116]. Both of these interactions rely on the isopre-
nylation of Rac. Therefore, reduction in ROS is likely a
consequence of statin inhibition of Rac prenylation result-
ing in the inability of Rac to interact effectively to form the
NADPH oxidase complex or RhoGDI.
The use of statins as a potential treatment for AD has
received substantial attention based on two epidemiolog-
ical studies that showed an association between statin use
and a reduced incidence of AD. These studies demon-
strated a ~70% reduction in AD incidence among patient
populations receiving statin treatment [117,118]. Despite
these promising findings, the use of statins in prospective
clinical trials has yielded unsatisfactory results, failing to
dramatically improve cognitive function or reduce serum

plasma concentrations of Aβ
40
or Aβ
42
[119,120]. Statins
remain an important tool to delineate the proinflamma-
tory effects of fibrillar Aβ peptides in vitro; however, the
efficacy of statins as a treatment for AD remains to be
defined by large well-controlled clinical trials currently
underway.
Conclusion
Alzheimer's disease is characterized by a variety of proin-
flammatory responses that act in concert to promote the
progressive pathophysiology associated with this disease.
The results summarized in this review suggest that ROS
and iNOS released through NADPH-dependent mecha-
nisms contribute to the extensive oxidative damage found
in the brains of AD patients. A greater understanding of
the intracellular signaling events that give rise to pro-
longed inflammatory responses may facilitate the discov-
ery of therapeutic agents that can ameliorate the prognosis
for AD patients. Indeed, clinical studies have addresses the
use of antioxidant and statins as potential therapies for
the treatment of AD, unfortunately these early studies
have yielded limited results. However, a disruption of oxi-
dative damage through the sustained inhibition of the
NADPH oxidase could possibly lead to an attenuation of
the neuronal cell loss induced by fibrillar Aβ peptides.
Journal of Neuroinflammation 2006, 3:30 />Page 9 of 12
(page number not for citation purposes)

Competing interests
The author(s) declare that they have no competing inter-
ests.
Authors' contributions
The authors contributed equally to the writing and editing
of this manuscript.
Acknowledgements
This work was supported by grants from the National Institutes of Health
(AG16740), the Blanchette Hooker Rockefeller Foundation, and the Amer-
ican Health Assistance Foundation (G.E.L.). B.L.W. was supported in part
through a Ruth L. Kirschstein National Research Service Award from the
National Institutes of Health (F32 AG24031).
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